title: Giant unilamellar vesicles (GUVs) as a new tool for analysis of caspase-8/Bid-FL complex binding to cardiolipin and its functional activity.
abstract: Cardiolipin (CL) has recently been shown to be both an anchor and an essential activating platform for caspase-8 on mitochondria. These platforms may be at the mitochondrial contact sites in which truncated Bid (tBid) has been demonstrated to be located. A possible role for CL is to anchor caspase-8 at contact sites (between inner and outer membranes), facilitating its self-activation, Bid-full length (FL) cleavage, tBid generation (and Bax/Bak activation and oligomerization), mitochondrial destabilization and apoptosis. We have developed an in vitro system that mimics the mitochondrial membrane contact site platform. This system involves reconstituting caspase-8, Bid-FL and CL complexes in giant unilamellar vesicles (GUVs). We first validated the system by flow cytometry analysis of light-scattering properties and nonyl acridine orange staining of their CL content. Then, we used flow cytometry analysis to detect the binding of active caspase-8 to CL and the subsequent truncation of bound Bid-FL. The tBid generated interacts with CL and induces GUV breakage and partial re-vesiculation at a smaller size. Our findings suggest an active role for mitochondrial membrane lipids, particularly CL, in binding active caspase-8 and providing a docking site for Bid-FL. This phenomenon was previously only poorly documented and substantially underestimated.
authors.affiliation: Institut Cochin, CNRS UMR, INSERM U, Université Paris V - R. Descartes, France.
authors.forename: O
authors.initials: O
authors.lastname: Jalmar
authors.forename: A J
authors.initials: AJ
authors.lastname: García-Sáez
authors.forename: L
authors.initials: L
authors.lastname: Berland
authors.forename: F
authors.initials: F
authors.lastname: Gonzalvez
authors.forename: P X
authors.initials: PX
authors.lastname: Petit
mesh_terms: D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism* / Q000502:physiology; D002308:Cardiolipins / Q000378:metabolism* / Q000502:physiology; D053181:Caspase 8 / Q000378:metabolism* / Q000502:physiology; D005434:Flow Cytometry; D006801:Humans; D008563:Membrane Lipids / Q000378:metabolism / Q000502:physiology; D008928:Mitochondria / Q000378:metabolism; D053835:Unilamellar Liposomes / Q000378:metabolism*
chemical_list: D051023:BH3 Interacting Domain Death Agonist Protein; D002308:Cardiolipins; D008563:Membrane Lipids; D053835:Unilamellar Liposomes; D053181:Caspase 8